Gene carriers with the use of polysaccharide and process for producing the same

ABSTRACT

Disclosed are gene carriers utilizing β-1,3-glucan and methods of preparing the same. The β-1,3-glucan has at least one 1,6-glucopyranoside branch and is chemically modified through periodate oxidation and reductive amination so as to impart nucleic acid-binding functional groups (for example, cationic functional groups) to at least some of the 1,6-glucopyranoside branches thereof. Triple helix β-1,3-glucan is dissolved in a polar organic solvent to form single-stranded β-1,3-glucan. By replacing, in the presence of a nucleic acid, the polar organic solvent for the solution containing the chemically modified single-stranded β-1,3-glucan by water, a complex (gene carrier) is formed in which the nucleic acid is bound to double-stranded β-1,3-glucan.

This application is a national stage application of PCT/JP02/02228,filedMar. 11, 2002, which claims priority to foreign applicationsJP2001-069655, filed Mar. 13, 2001, and JP2001-130705, filed Apr. 27,2001.

TECHNICAL FIELD

The present invention relates to a gene carrier composed of a complex ofa polysaccharide combined with a nucleic acid, and more particularly toa novel artificial material (compound) which is capable of interactingwith a nucleic acid to form a complex with the nucleic acid for carryingthe nucleic acid for use as an antisense agent and other applications.

BACKGROUND ART

The analysis of the human genome is expected to be completed in theearly twenty-first century. For effective utilization of the outcome ofthe analysis, it is indispensable to develop a new technology forartificial manipulation of nucleic acids (carrying, sequencerecognition, and control of transcription or translation of nucleicacids). The most important material for manipulating nucleic acids isconsidered to be a carrier capable of interacting with a nucleic acidsuch as DNA to support or carry the nucleic acid. However, in-vivo usesof conventional gene carriers composed of artificial materials haveproduced no significant results in human clinical studies. This can beattributed especially to (1) low gene-transferring efficiency, (2)difficulty in controlling the association and dissociation of genes(Cotton et al., Meth. Enzymol. 217: 618-644 (1993)); and (3) cytotoxitycaused by cationic carrier materials (Choksakulnimitr et al., J.Control. Rel., 34: 233-241 (1995)).

Although viruses such as retroviruses (Miller, Nature 357: 455-460(1992)) or adenoviruses (Mulligan, Science 260: 926-932 (1993)) haveshown very promising in-vitro results as gene carriers, in-vivo use ofthese naturally occurring materials is restricted, especially because ofinflammatory action, immunogenetic properties or the risk of integrationinto the genome or mutagenesis induction due to the viruses (Crystal,Science 270: 404-410 (1995)). Thus, as a substitute for suchnaturally-originating gene vectors, there has been proposed use ofnon-viral carriers composed of an artificial material which can behandled in an easier manner and can carry DNAs into the cells in a moreefficient manner as compared with the viruses (Tomlinson and Rolland, J.Contr. Rel., 39: 357-372 (1996)).

At present, it is polyethyleneimine (PEI) that is the most extensivelystudied non-viral, artificial carrier material. It has been shown thatPEI, a cationic polymer assuming a three dimensional branched structure,may result in transfection in a considerably highly efficient manner forvarious adhesive and floating cells, (Boussif et al., Gene Therapy 3:1074-1080 (1996)). For example, 95% in-vitro transfection wasaccomplished in the 3T3 fibroblast cell line. In-vivo gene transfectioninto mouse brain using PEI as a carrier resulted in long-termexpressions of the reporter gene and Bcl 2 gene in the neuron and theglial cell, the results being comparable to those obtained with the genetransfection using the adenovirus (Abdallah et al., Hum. Gene Ther. 7:1947-1954 (1996)).

However, the safety of cationic polymers such as polyethylimine has notyet been established. While the introduction of amino groups isindispensable for imparting a cationic charge to such a polymer, anamino group has a risk of toxicity in the living body due to its highphysiological activity. In fact, no cationic polymers studied so farhave yet been put into practice, or yet been registered in the“Pharmaceutical Additives Handbook” (edited by the PharmaceuticalAdditives Association of Japan and published by YakujinipposhaPublishing Co.).

β-1,3-glucan is a polysaccharide which has been clinically put topractical use as an intramuscular injection. It has been known since along time ago that the polysaccharide takes, as it occurs naturally, atriple-stranded helix structure (cf., for example, Theresa M. McIntire,David A. Brant, J. Am. Chem. Soc., Vol. 120, 6909 (1998)). Thispolysaccharide has already been confirmed with respect to its in-vivosafety, based on the results of practical use for some 20 years as anintramuscular injection (Simizu et al., Biotherapy, Vol. 4, 1390 (1990);Hasegawa, Oncology and Chemotherapy, Vol. 8, 225 (1992)).

PCT/US95/14800 teaches that β-1,3-glucan is chemically modified so as tobe capable of forming a conjugate with a bioactive material such as DNA,for use as a gene carrier. However, this prior art describes nothing buta preparation of a β-1,3-glucan/bioactive material conjugate wherein theβ-1,3-glucan is utilized as it occurs naturally, i.e. in the form of atriple-stranded helix structure, to form the conjugate through covalentcoupling.

Recently, the present inventors and others have found that β-1,3-glucancan form, as it is artificially treated, a new type of complex with anucleic acid (PCT/JP00/07875; Sakurai and Shinkai, J. Am. Chem. Soc.,122, 4520 (2000); Kimura, Koumoto, Sakurai and Shinkai, Chem. Lett.,1242 (2000)). More specifically, it has been found that thetriple-stranded helix form, which the polysaccharide takes as itnaturally occurs, can be unbound into separate single strands bydissolving the triple helix (triple-stranded helix) polysaccharide in apolar solvent. Thus, as the solution is added with a single-strandednucleic acid and the solvent is replaced by water (as a renaturationprocess), there is formed a triple-stranded helix complex composed of asingle-stranded nucleic acid and a double-stranded polysaccharide. Thebinding between the polysaccharide and the nucleic acid in the complexis considered to be primarily through hydrogen bonding. (K. Sakurai, R.Iguchi, T. Kimura, K. Koumoto, M. Mizu, and S. Shinkai, Polym.Preprints, Jpn., 49, 4054 (2000)). The energy of this binding isrelatively small, resulting in an easy dissociation of the complex. Itis necessary to render this complex more strongly affinitive withnucleic acids in order to utilize the complex as a gene carrier.

It is an object of the present invention to provide a new type of genecarrier which is capable of interacting with and bonding to a nucleicacid such as DNA and RNA, without destroying the nucleic acid, to form awater-soluble complex so as to be applicable under biologicalconditions, and which is also capable of dissociating the nucleic acidfrom the complex and rebonding to such nucleic acid when necessary.

DISCLOSURE OF THE INVENTION

The present inventors discovered, through extensive studies foraccomplishing the above-mentioned object and that led to the presentinvention, that introduction of nucleic acid-binding functional groupsinto β-1,3-glucan can make the polysaccharide highly interactive with anucleic acid such as DNA and RNA so as to form a nucleic acid-polymercomplex which is suitable for use in carrying genes, the separation ofnucleic acids, or the controlling of the transcription or translation.

Thus, according to the present invention there is provided a method ofpreparing a gene carrier composed of a complex in which asingle-stranded nucleic acid is bound to a double-stranded β-1,3-glucanwherein the β-1,3-glucan has at least one 1,6-glucopyranoside branch perrepeating unit of polysaccharide, comprising the steps of (i) chemicallymodifying β-1,3-glucan so as to impart nucleic acid-binding functionalgroups to at least some of the 1,6-glucopyranoside branches thereof,(ii) dissolving triple helix β-1,3-glucan in a polar organic solvent toform single-stranded β-1,3-glucan, and (iii) replacing, in the presenceof a nucleic acid, the polar organic solvent for the solution containingthe single-stranded β-1,3-glucan by water so as to form the complex inwhich the single-stranded nucleic acid is bound to the double-strandedβ-1,3-glucan.

According to the present invention there is also provided a gene carriercomposed of a complex in which a single-stranded nucleic acid is boundto a double-stranded β-1,3-glucan wherein the β-1,3-glucan has at leastone 1,6-glucopyranoside branch per repeating unit of polysaccharide, andwherein at least some of the 1,6-glucopyranoside branches are impartedwith nucleic acid-binding functional groups. In a preferred embodimentof the present invention the nucleic acid-binding functional group is acationic functional group, a steroid-based functional group, an aminoacid-based functional group, or an intercalator-based functional group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows a process of preparing the gene carrier ofthe present invention in comparison with that of the prior art.

FIG. 2 illustrates a reaction process by which the branch ofβ-1,3-glucan is subjected to a periodate oxidation in accordance withthe present invention.

FIG. 3 illustrates a process by which the branch of β-1,3-glucan isimparted with cationic functional groups to yield the cationicallymodified polysaccharide in accordance with the present invention.

FIG. 4 illustrates a process of synthesizing a compound providing asteroid-based functional group for use in the present invention.

FIG. 5 illustrates a process by which an amino acid-based functionalgroup is imparted to the branch of β-1,3-glucan.

FIG. 6 illustrates a process of synthesizing an acridine derivativehaving a spacer for use as an intercalator-based functional group in thepresent invention.

FIG. 7 illustrates the results of a UV absorption experiment forstoichiometrically evaluating the complex of the present invention.

FIG. 8 shows a summary of CD spectra of the complexes of amino group(cationic functional group)-modified schizophyllan with poly(C) as wellas those of unmodified schizophyllan or polyethyleneimine with poly(C)for comparison.

FIG. 9 illustrates temperature dependency of the CD spectra of thecomplexes of the amino group-modified schizophyllan and poly(C) as wellas those of unmodified schizophyllan and poly(C) for comparison.

FIG. 10 illustrates the results of experiments for evaluating antisenseeffect by the modified schizophyllan-antisense DNA complex according tothe present invention.

FIG. 11 is a representation of experimental results showing the changewith the elapse of time in the function of the modified schizophyllan asa gene carrier according to the present invention.

FIG. 12 is a representation of experimental results showing that thenucleic acid-polysaccharide complex of the present invention isresistant to nucleotides.

FIG. 13 is a graphical representation showing that the nucleicacid-polysaccharide complex of the present invention is resistant tonuclease resulting in increased antisensing effects.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, β-1,3-glucan having a triple helixstructure as it naturally occurs is artificially treated to provide anew type of gene carrier composed of a complex in which a target nucleicacid (DNA, RNA) is bound to double-stranded β-1,3-glucan.

FIG. 1 schematically shows the principle of the present invention incomparison with the prior art as described in PCT/US/9514800. FIG. 1(A)illustrates the gene carrier as described in PCT/US95/14800, in whichtriple helix β-1,3-glucan is chemically modified as it naturally occurs,and is bound with a target bioactive material such as an antisense DNAthrough hydrolysable covalent bonds. FIG. 1(B) relates to the ideaconceived by the present inventors, in which triple helix β-1,3-glucanis dissolved in a polar organic solvent to form a single-strandedpolysaccharide. (I) illustrates a complex disclosed in an earlier patentapplication by the present inventors (PCT/JP00/07875), in which thesolvent for the solution containing the single-stranded β-1,3-glucan isdirectly replaced by water to form the nucleic acid-polysaccharide(double-stranded) complex. The binding energy for this complex isprimarily due to hydrogen bonding and thus relatively small, which maylead to the problem of dissociation between the nucleic acid and thepolysaccharide. This drawback is overcome by the process according tothe present invention as illustrated in (II): A complex in which atarget nucleic acid is bound to double-stranded β-1,3-glucan is formedby chemically modifying the branches (the side chains) of β-1,3-glucanto impart nucleic acid-binding functional groups thereto, subsequentlydissolving the resultant β-1,3-glucan in a polar solvent, and finallyreplacing the solvent by water. The binding strength for this complex isdue to the interactive actions (for example, ionic bonding) between thenucleic acid-binding functional group and the nucleic acid and thereforeenhanced as compared with that of the complex shown in (I).

Thus, according to the present invention, β-1,3-glucan is once renderedinto single-stranded form. The polysaccharide is then subjected to arenaturation process in water for forming double-stranded β-1,3-glucanduring which process there emerge hydrogen-bonding sites within thepolysaccharide. In the complex of the present invention, a nucleic acidis appropriately bound to the double-stranded β-1,3-glucan through thehydrogen-bonding sites and the nucleic acid-binding functional groups,and the nucleic acid may be released from the complex in a subsequentprocess (for example, the introduction of the complex into in-vivo orcultured tissue). By contrast, in the complex as described inPCT/US95/14800, a bioactive material such as nucleic acid is bond totriple helix β-1,3-glucan (in which there are no vacant hydrogen-bondingsites because the triple helix structure is constructed through thehydrogen-bonds) only through hydrolysable covalent bonds. The bioactivematerial can be released from the complex only under the conditionswhere hydrolysis occurs.

β-1,3-glucan is a generic term for polysaccharides in which glucoserings are linked by β-bonds through hydroxyl groups at the positions 1and 3. The β-1,3-glucan to be used in the present invention is one whichhas at least one 1,6-glucopyranoside branch per repeating unit ofpolysaccharide. Examples of β-1,3-glucan having such structure for usein the present invention include, but are not limited to, schizophyllan,lentinan, pachyman, grifolan and sucleroglucan. β-1,3-glucan for use inthe present invention may include a compound derived from chemicaltreatment of a natural β-1,3-glucan and having more than 10%, preferablymore then 20% by weight, of the above-defined repeating units. Suchβ-1,3-glucan for use in the present invention is sometimes referred toherein to as “the polysaccharide of the present invention.”

The “nucleic acid-binding functional group,” as used herein, to beimparted to the 1,6-glucopyranoside branches of β-1,3-glucan for thepresent invention, refers to a functional group or atomic group whichinteracts with a nucleic acid so as to enhance binding (affinity)between the polysaccharide of the present invention and the nucleicacid. Suitable nucleic acid-binding functional groups for use in thepresent invention include cationic functional groups, steroid-basedfunctional groups, amino acid-based functional groups andintecalator-based functional groups. Cationic functional groups impartedto the branches of the polysaccharide of the present invention canenhance the binding between the polysaccharide and the nucleic acidowing to electrostatic interaction with the negative charges on thenucleic acid such as DNA or RNA. Steroid-based functional groups oramino acid-based functional groups enhance the binding between thepolysaccharide and nucleic acid, owing to hydrophobic interaction withthe nucleic acid. Intercalator-based functional groups intercalatebetween the base-pairs of DNA or RNA and enhance the binding between theDNA or RNA and polysaccharide, owing to hydrogen bonding or hydrophobicinteraction.

Cationic functional groups, particularly amino groups, may exhibittoxicity because of their physiological activity, depending upon thequantity of the functional groups. Amino groups are present innumerablyin living organisms and an amino group is not itself toxic. The presenceof an amino group in every repeating unit of polymer may often exhibittoxicity, as in polyethyleneimine which has been conventionally studiedas an artificial carrier. The present invention features decreasedintroduction of nucleic acid-binding functional groups typified bycationic functional groups into the branches of β-1,3-glucan, so as tomaintain the in-vivo safety of the polysaccharide to the utmost.Depending upon the types of nucleic acid-binding functional groups, therate of introduction of nucleic acid-binding functional groups isdetermined based on three parameters: the toxicity, the releasabilityand the safety of the complex. The nucleic acid releasability and thecomplex stability depend upon the sequence and the number of bases ofthe target DNA In the present invention nucleic acid-binding functionalgroups are introduced generally at a rate of 50% or less, preferably 30%or less, more preferably 15% or less, and the most preferably 10% orless, based on the mole % of the repeating unit of the polysaccharide ofthe present invention.

It is generally known that the introduction of cationic functionalgroups into polyethyleneimine (PEI) at a rate of 10% or less will resultin loss in the stability of PEI-nucleic acid complex. This is becausethe complex is formed only by electrostatic forces between the ionpairs. In the present invention the formation of the complex isessentially due to the hydrogen-bonding (plus the hydrophobicinteraction) between the polysaccharide and the nucleic acid, while thebinding between the nucleic acid-binding functional group and thenucleic acid serves as a trigger for the formation of the complex. Thisis evidenced from the fact that the CD spectrum of the complex of thepolysaccharide of the present invention chemically modified withcationic functional groups and the nucleic acid is not different fromthat of the complex of the unmodified polysaccharide and the nucleicacid, as shown in Example 3 and Comparative Example 2. By contrast, inthe case of polyethyleneimine, the complex therefrom exhibits a highlydecreased CD spectrum in which there is observed no conformationresulting from the nucleic acid, as shown in Comparative Example 4.Thus, the gene carrier of the present invention, in which β-1,3-glucanis imparted with nucleic acid-binding functional groups on its branches,is essentially different from the conventional polycationic vector.

While the molecular weight of β-1,3-glucan for use in the presentinvention may vary depending upon the specific purpose of use, it ispreferably 2000 or more in terms of weight-average molecular weight. Apolysaccharide having a too-low molecular weight will not work as apolymer and make formation of the complex difficult.

A characteristic feature of the method for preparing the complex for useas a gene carrier according to the present invention is that it includesa step of forming a single-stranded polysaccharide, by dissolving triplehelix β-1,3-glucan having the above-defined branches in a polar solvent.While any polar organic solvent may be used so long as it can unbind thetriple helix, preferred examples include dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, propylene carbonate, methylene carbonate,and sulfolane, in which dimethyl sulfoxide and dimethyl formamide aremore preferred. While the triple helix can be unbound simply bydissolving β-1,3-glucan for use in the present invention in such polarorganic solvent, a heating procedure may be added if necessary. Theunbinding of the triple helix may be carried out by dissolving it in analkaline solution having a pH of 10 or higher, preferably 12 or higher,depending upon the specific purpose of use. Alternatively, a neutralaqueous solution containing β-1,3-glucan may be heated at a temperatureof 100° C. or higher, preferably 120° C. or higher in an autoclave. Theunbinding of the triple helix into the single strands can be ascertainedby measuring the molecular weight using GPC (gel permeationchromatography), the light scattering method or the sedimentationequilibrium method in order to determine that the molecular weight hasbecome one-third owing to unbinding.

A further characteristic feature of the method for preparing the complexfor use as a gene carrier of the present invention is that it includesthe step of chemically modifying the polysaccharide of the presentinvention, i.e. β-1,3-glucan having at least one 1,6-glucopyranosidebranch per repeating unit of polysaccharide, so as to impart theabove-defined nucleic acid-binding functional groups to at least some ofsaid 1,6-glucopyranoside branches.

Any ordinary method known in the field of organic chemistry can beemployed for chemically modifying the polysaccharide of the presentinvention so as to impart nucleic acid-binding functional groupsthereto. For example, a general organic reaction may be applied, such asWilliamson reaction or addition reaction to hydroxyl groups, additionreaction or condensation reaction to aldehydes or carboxylic acidsproduced by the oxidation of hydroxyl groups, or Williamson reactionfollowing the activation (e.g. halogenation, tosylation or mesylation)of hydroxyl groups. Reductive amination onto the reducing terminal ofthe saccharide chain or aminolysis of lactone produced by the oxidationof the reducing terminal may also be applied.

Of these methods, the most preferred comprises periodate-oxidizing the1,6-glucopyranoside branches of the polysaccharide of the presentinvention and then reductive-aminating the periodated1,6-glucopyranoside branches to impart nucleic acid-binding functionalgroups. The periodate-oxidation and the reductive-amination may beconducted on the single-stranded 6-1,3-glucan formed by dissolving thenatural polysaccharide in a polar organic solvent as explained earlierwith reference to FIG. 1. Alternatively, the periodate-oxidation may beconducted on the triple helix β-1,3-glucan while the reductive-aminationmay be carried out on the single-stranded β-1,3-glucan. Stillalternatively, both the periodate-oxidation and the reductive-aminationmay be conducted on the triple helix β-1,3-glucan.

Periodate oxidation, as used herein, is a reaction that converts a1,2-diol quantitatively to the corresponding aldehyde, in which anyalkali metal periodate can be used as the reagent, including sodiumperiodate, potassium periodate and rubidium periodate. Of suchperiodates, sodium periodate is most preferably used from the viewpointof solubility and cost. Any polar solvent can be used for the reactionwhen the reaction is applied to the polysaccharide of the presentinvention, so long as it can dissolve the polysaccharide and does notadversely affect the reaction. In this respect, water is preferable.

When the periodate oxidation is conducted on single-strandedβ-1,3-glucan formed by dissolving the starting polysaccharide in a polarorganic solvent, it is convenient to use as a solvent for the periodateoxidation the same solvent as that used in the process for forming thesingle-stranded polysaccharide, dimethyl sulfoxide being particularlypreferred. While the temperature for the periodate oxidation is notparticularly limited unless the autodecomposition of the periodateshould proceed, the reaction is generally carried out at a temperaturein the range of 0 to 50° C.

Thus, as illustrated in FIG. 2, the bond between the 3- and 4-positionsor between the 2- and 3-positions in the 1,6-glucopyranoside branch ofβ-1,3-glucan is cleaved to convert the hydroxyl group at these positionsto aldehyde groups. As mentioned previously, a periodate oxidationfeatures a selective reaction onto the C—C bond between diols at the 1-and 2-positions. Since β-1,3-glucan contains no 1,2-diols within itsbackbone, in the process of the reaction according to the presentinvention, there occurs no cleavage or modification on the backbone ofthe polysaccharide.

The β-1,3-glucan having the aldehyde groups within its branches asprepared above is then subjected to a reductive amination so as toimpart nucleic acid-binding functional groups to the branches. The“reductive amination”, as used herein, refers to a reaction in which acondensation reaction between an aldehyde and an amino group is followedby a reduction, as a measure for introducing a functional group througha covalent bond. While this reaction can be applied to any compoundhaving a primary or secondary amine moiety or a hydrazine moiety, it ispreferable to avoid a compound having a functional group, which may beaffected by the reduction reaction, for example, vinylketone. A reducingagent for the reduction reaction can be selected from among sodiumborohydride, lithium borohydride and sodium hydrocyanide. While anysolvent can be used for the reaction when the reaction is applied to thepolysaccharide of the present invention so long as it can dissolve thepolysaccharide and does not adversely affect the reaction, the mostpreferred is water or dimethyl sulfoxide.

Thus, in the present invention, the 1,6-glucopyranoside branches, whichhave been imparted with nucleic acid-binding functional groups throughthe chemical modification comprising the periodate oxidation and thereductive amination, can be expressed by the following general formula(1). In formula (1), the two X's are generally identical nucleicacid-binding functional groups, but may be different nucleicacid-binding functional groups.

As will be understood from the foregoing, the nucleic-acid bindingfunctional groups to be imparted to the 1,6-glucopyranoside branches ofβ-1,3-glucan by the reductive amination following the periodateoxidation according to the preferred embodiment of the present inventionare those derived from a compound which contains a primary or secondaryamine moiety or a hydrazine moiety to which the reductive amination canbe applied.

Examples of the cationic functional groups suitable for use in thepresent invention include, but are not limited to, those derived fromthe following chain or cyclic compounds which contain at least oneprimary or secondary amino group. These compounds can be easilysynthesized from commercially available compounds containing an aminogroup or groups.

FIG. 3 illustrates an example of a process for preparing the chemicallymodified polysaccharide, by imparting to the branch the cationicfunctional groups as the nucleic acid-binding functional groupsaccording to the present invention. In the figure, (i) denotes the stepof the oxidation with a periodate, (ii) denotes the step of theformation of a Schiff base, and (iii) denotes the step of the reductionof the Schiff base with sodium borohydride. In the case of β-1,3-glucanhaving branches containing an unreacted hydroxyl group at the3-position, there are obtained products as expressed by 2.X, 3.X, and4.X. The reactions occur at the branch or side chain as shown by 5 and 6in the figure.

The steroid-based functional groups to be imparted to the branches ofthe polysaccharide of the present invention by the reductive aminationfollowing the periodate oxidation are preferably those derived from thecompounds expressed by the following formula (2):

In the formula, R₁, R₂ and R₃ independently express hydrogen atom or asubstituent containing carbon, oxygen, nitrogen and/or hydrogen atom(s).R₄ denotes a spacer moiety which is hydrogen atom or is derived from achain or cyclic compound containing carbon, oxygen, nitrogen and/orhydrogen atom(s). The atomic group containing R₄ may be bound to anyposition of the steroid ring. A compound falling outside theabove-mentioned general formula (1) may also be used, provided that itcontains a steroid ring, as exemplified by a corticosterone or cortisolderivative.

Such compounds may be prepared in accordance with a reaction scheme asshown in FIG. 4 in which the hydroxyl group bound to the steroid ring iscaused to react with a diamine. The compound is then subjected to thereductive amination as mentioned earlier to impart the steroid-basedfunctional groups to the branches of the β-1,3-glucan.

The amino acid-based functional groups to be imparted to the branches ofthe β-1,3-glucan by the periodate oxidation and the reductive aminationaccording to the preferred embodiment of the present invention are thosederived from an amino acid as expressed by the following general formula(3) or from a peptide composed of a plurality of such amino acids:

In the formula, R₅ denotes the side chain of amino acid, including CH₃(in the case of alanine), CH₂Ph (in the case of phenylalanine, wherin Phdenotes a phenyl group), (CH₃)₂CH (in the case of valine), (CH₃)₂CHCH₂(in the case of leucine), CH₂OH (in the case of serine), CH₃(OH)CH (inthe case of threonine), CH₂—SH (in the case of cysteine) and so on.

The amino acid-based functional groups derived from an amino acid asexpressed by the formula (3) are introduced into the branches ofβ-1,3-glucan in accordance with a reaction scheme as shown in FIG. 5, inwhich the amino group of the amino acid is utilized for the introductionfollowing the esterification of the amino acid. Without theesterification of the amino acid, the reductive amination would not bepossible because an amino acid usually behaves as an amphoteric ion. Inthe case of lysine, arginine, aspartic acid or glutamic acid, it is alsonecessary to protect the side chain of the amino acid. The protection ofamino acid side chain may be carried out in any manner known in thefield of amino acid chemistry, for example, with reference to“Experimental Chemistry Series” (Maruzen Co.) or “BiochemicalExperiments Series” (Tokyo-Kagaku-Dojin Co.).

The type of amino acid bound to the polysaccharide may vary dependingupon the purpose of use. For example, for the purpose of enhancing theaffinity with a nucleic acid due to the formation of an ion-pair, it ispreferred to use a basic amino acid. For enhancing the transfectionefficiency, it is preferred to use a hydrophobic amino acid such asarginine. While FIG. 5 illustrates the case where the N-terminus isbound directly to the branch (the side chain) of β-1,3-glucan, as amatter of convenience, there may be used an appropriate spacer moietytherebetween if necessary. The amino acid-based functional groups foruse in the present invention may be derived from a peptide composed of aplurality of amino acids. Any peptide capable of interactingspecifically with a nucleic acid may be used, including fibronectin,proteins selected from among the integrin family, integrin-bindingpeptides (cystein-tyrosine-glycine-glycine-arginine-glycine-asparticacid-threonine-proline), DNA-interacting—peptide-complexed nucleic acid(PNA) and so on. They can be introduced into the branches of thepolysaccharide in a similar manner as in the case of amino acidmentioned above.

The intercalator-based nucleic acid-binding functional groups to beimparted to the branches of the polysaccharide of the present inventionby the reductive amination following the periodate oxidation inaccordance with the preferred embodiment of the present invention arethose from intercalating compounds containing an amino group or acarboxyl group. Preferred examples of such compounds include thosecontaining an acridine, proflavin or ethidium moiety as expressed by thefollowing formulae (4), (5), and (6), respectively.

While it is possible to bind these compounds having an amino group, suchas expressed by the formulae above, to the branches (the side chains) ofthe polysaccharide of the present invention by the reductive aminationfollowing the periodate oxidation, it is preferred for the compounds tohave a spacer moiety so that they will be arranged at an appropriatedistance from a nucleic acid such as DNA in interacting with it. Aconvenient method is to introduce an aminoalkane carboxylate through anamide bond with an appropriate condensation agent (for example,dicyclohexyl carbodiimide, cf. Experimental Chemistry Series, MaruzenCo.) FIG. 6 illustrates a process for the introduction of such a spacermoiety, acridine, as an example. The resultant is bound to the branchesof β-1,3-glucan of the present invention by the reductive aminationexplained previously. When an intercalating compound containing acarboxyl group is used, a diamine having an appropriate spacer moiety isintroduced with a condensation agent, in a manner similar to theaforesaid. The resultant is then bound to the branches of thepolysaccharide of the present invention by the reductive amination.

The third characteristic feature of the method for preparing the genecarrier of the present invention is that it includes the step ofreplacing, in the presence of a nucleic acid, the polar organic solventfor the solution containing the single-stranded β-1,3-glucan, followingthe chemical modification, by water so as to form the complex in whichthe nucleic acid is bound to the double-stranded β-1,3-glucan. Morespecifically, in the process where the β-1,3-glucan taking thesingle-stranded form in the polar organic solvent will revert in waterto the triple-stranded form (renaturation process), the presence of anucleic acid results in the formation of a new complex composed ofsingle-stranded nucleic acid plus double-stranded polysaccharide. Whilesuch regeneration process can be carried out by replacing the solventwith water generally through dialysis or ultrafiltration, simpledilution of the polar organic solvent with water will also work. It isfurther to be noted, as can be seen from the foregoing, that thereplacement process of the polar organic solvent by water may be appliedto any of single-stranded β-1,3-glucan obtained by the chemicalmodification comprising the periodate oxidation of the triple helixβ-1,3-glucan, followed by the dissolution in the polar solvent and thenthe reductive amination; single-stranded β-1,3-glucan obtained by thechemical modification comprising dissolving the triple helixβ-1,3-glucan in the polar organic solvent to prepare the single-strandedform, followed by the periodate oxidation and the reductive amination;and single-stranded β-1,3-glucan obtained by the chemical modificationcomprising the periodate oxidation and the reductive amination of thetriple helix β-1,3-glucan, followed by the dissolution in the polarorganic solvent.

The polysaccharide of the present invention thus prepared with thebranches having been imparted with nucleic acid-binding functionalgroups is capable of interacting with a nucleic acid such as DNA or RNAand forming a complex with the nucleic acid. The formation of thecomplex can be confirmed by studying the conformational change, forexample, by measuring the CD (circular dichroism) spectra.

The complex of the present invention composed of the double-strandedβ-1,3-glucan and the nucleic acid bound to the branches of thepolysaccharide through the nucleic acid-binding functional groups isusually water-soluble, and may undergo a dissociation and a rebindingdue to temperature change and/or pH change. The binding capabilitythereof varies depending upon the chemical structure of the DNA or RNA(A, T, G, C, U). The complex has resistance to nuclease, so that nodecomposition of the nucleic acids (genes) occurs. Thus, the complex ofthe present invention can be used as a gene carrier to manipulatevarious types of genes. A target gene (DNA or RNA), for example, anantisense nucleic acid, can be carried by the gene carrier of thepresent invention, in the form of the complex in which the nucleic acidis bound to the β-1,3-glucan, and introduced into in-vivo or culturedtissues. The present invention is therefore expected to contribute tothe development of medicines and reagents for diagnostic and therapeuticpurposes. The complex of the present invention may also be utilized as anucleic acid-protecting agent since it has resistance to nuclease.

Various types of nucleic acids can be used in the preparation of thecomplex according to the present invention. For example, any antisensenucleic acid as described in “Idenshi-chiryo (gene therapy) R&DHandbook, N.T.S.” is usable. Besides deoxyribonucleic acid, there may beused such nucleic acid as phosphorothioate nucleic acid,phosphoroamidate nucleic acid, peptide nucleic acid, purine ribose, andmethoxy-ethoxy DNA. If necessary, the present invention can be appliedto double-stranded DNA, in which the DNA is believed to bind partly tothe polysaccharide of the present invention through the single-strandedregion as in the bulge or bulge loop, although the detailed mechanismhas not yet been elucidated.

EXAMPLES

The invention will be more fully described with reference to thefollowing examples and comparative examples, which are only forexemplifying purposes and not for restricting the invention.

Example 1 shows a case where schizophyllan, a typical polysaccharide ofthe present invention, having a molecular weight of 150000, is impartedwith cationic functional groups as the nucleic acid-binding functionalgroups. Example 2 set out a characterization (the molecular weight andthe rate of introduction of the functional groups) of the cationicpolysaccharide thus obtained. Example 3 and Comparative Examples 1 & 2relate to measurements of CD spectra of complexes of the cationicpolysaccharide with poly(C), a single-stranded RNA, demonstrating thatthe chemical modification according to the present invention resulted inno change in CD spectrum. Example 4 describes melting behaviors of thecomplexes, demonstrating the increased stability of the cationicallymodified polysaccharide based complex. Example 5 describes formation ofcomplexes of the cationically modified polysaccharide, as compared withunmodified polysaccharide, with homogeneous nucleic acid other thanpoly(C), and an antisense DNA, a heterogeneous nucleic acid. Example 6sets forth an experimental example to demonstrate that the gene carrierof the present invention, composed of the modified polysaccharide(schizophyllan) serves to carry an antisense DNA, thereby regulating theexpression of a protein. Example 7 describes change in the regulation ofthe protein expression by the gene carrier for the antisense DNA as setforth in Example 6, with the elapse of time. Example 8 shows an exampleof an experiment for studying the resistance of the polysaccharide to anuclease. Example 9 describes an experiment by which it is demonstratedthat the carrier composed of the modified polysaccharide (schizophyllan)of the present invention serves to regulate the expression of a proteinand is resistant to a nuclease. Example 10 is to show that the modifiedschizophyllan of the present invention is capable of forming a complexwith a nucleic acid and the complex may undergo a dissociation in thepresence of a complementary strand.

Example 11 describes synthesis of a polysaccharide of the presentinvention, which is imparted with steroid-based functional groups as thenucleic acid-binding functional groups. Example 12 describes synthesisof another polysaccharide of the present invention, which is introducedwith peptides as the nucleic acid-binding functional groups. Example 13sets forth synthesis of a further polysaccharide of the presentinvention, which is imparted with intercalator-based functional groupsas the nucleic acid-binding functional groups. Example 14 is todemonstrate that the polysaccharide of the present invention as preparedin Example 11 is capable of forming complexes with a nucleic acid.Example 15 demonstrates that the polysaccharide of the present inventionas prepared in Example 12 is capable of forming a complex with a nucleicacid and the complex can be introduced into hepatic cells. Example 16 isto demonstrate that the polysaccharide of the present invention asprepared in Example 1 is capable of forming a complex with a nucleicacid and the complex can be introduced into cancer cells.

Example 1 Synthesis of Cationic Polysaccharide (Amino Group-ModifiedSchizophyllan), by Chemical Modification with Cationic Functional Groups

A polysaccharide of the present invention was synthesized in accordancewith the reaction scheme as shown FIG. 3. It is possible to regulate therate of introduction of amino group by regulating the equivalent numberof sodium periodate for the periodate oxidation. The same method ofsynthesis is applicable regardless of the rate of introduction. Thepresent example relates to the synthesis of cationic functionalgroup-modified schizophyllan in which the schizophyllan is introducedwith amino groups at a rate of introduction of approximately 2.5%, 17%or 37%. The amino group introduced was derived from 2-aminoethanol. Itis possible to regulate the rate of introduction of the amino group byregulating the equivalent number of the sodium periodate with respect tothe branching glucose moiety. The experimental results are shown inExample 2.

Triple helix schizophyllan was prepared in accordance with theconventional method as described in “A. C. S. 38(1), 253 (1997);Carbohydrate Research, 89, 121-135 (1981)): Schizophyllum commune. Fries(ATCC 44200) available from American Type Culture Collection wassubjected to a stationary culture in a minimal medium for seven days.After removal of the cellular materials and insoluble residues, thesupernatant was subjected to a supersonic treatment to yieldschizophyllan of a triple helix structure having a molecular weight of450000.

100 mg of the thus obtained schizophyllan was dissolved in 100 ml water.To the resultant solution was added slowly an aqueous solution of sodiumperiodate (in an equivalent of 4%, 40%, or 500% (an excess amount) basedon the branching glucose of the schizophyllan) and stirring wasperformed for two days at 4° C. The reaction solution was subjected todialysis through a membrane with an exclusion limit of 12000, followedby lypophilization. The white solid product was dissolved in 20 mldimethyl sulfoxide, a polar organic solvent. To the resultant solutionwas added 2 ml 2-aminoethanol (an excess amount: more than 10000equivalents) and then stirring was performed for two days at roomtemperature. Then, there was added 100 mg sodium borohydride, followedby stirring for one day at room temperature. After the excess sodiumborohydride was deactivated with acetic acid, reprecipitation withmethanol was conducted to yield the modified polysaccharide(schizophyllan) to which the cationic functional groups were imparted.

Example 2 Characterization of the Cationic Polysaccharide

Characterization of the polysaccharide imparted with the cationicfunctional groups as prepared in Example 1 was performed by measuringthe molecular weight and the rate of introduction of the amino group.The molecular weight was examined through gel permeation chromatography(GPC) and also by measuring the viscosity. The molecular weight of thepolysaccharide was found to be 150000, i.e., one third of that of thestarting triple helix schizophyllan. The rate of introduction of theamino group was determined on microanalysis of nitrogen by elementalanalysis (lower detection limit: 0.05%). The microanalysis of nitrogenwas performed three times for each sample, with the upper and lowervalues being shown in the following table.

TABLE 1 Periodate Equivalent Nitrogen Content Amino Group IntroductionRate  4% 0.09-0.12%  2.1-2.8% 40% 0.69-0.75% 16.3-17.8% 500%  1.15-1.54%35.2-37.4%

Example 3 Interaction of Amino Acid-Modified Schizophyllan with Poly(C)

The thus-prepared schizophyllans modified with 2.5%, 17% and 37% ofamino group (as the cationic functional group) were each dissolved indimethyl sulfoxide, a polar organic solvent, to give a finalconcentration of 0.5 g/dL. To each resultant solution 100 μl, there wereadded pure water 900 μl, 10 mM Tris buffer 100 μL, and 0.1 g/L poly(C)(Pharmacia) solution 100 μl. The resultant mixtures were all clear,homogeneous solutions.

Comparative Example 1 Interaction of Unmodified (Natural) Schizophyllanwith Poly(C)

A mixture was prepared in the same manner as in Example 3 usingunmodified schizophyllan. The resultant mixture was a clear, homogeneoussolution.

Comparative Example 2 Interaction of Polyethyleneimine with Poly(C)

A mixture was prepared in the same manner as in Example 3 usingpolyethyleneimine in place of schizophyllan. The resultant solution wasturbid. The solutions as prepared in Example 3 and Comparative Examples1 & 2 were each matured in a refrigerator overnight, and then subjectedto CD spectrum measurement on a circular dichroism apparatus (Jasco) toconfirm the formation of a complex.

FIG. 8 shows the CD spectrum data (at 5° C.) for Example 3 andComparative Examples 1 & 2 in comparison with a system in which nopolymeric material was present (i.e. only with poly(C)). The CD spectrumdata were expressed in terms of molecular ellipticity (cf. BiochemicalExperiments Series Vol. 2, Nucleic Acid Chemistry II, Edited byBiochemical Society of Japan, Tokyokagaku-Dojin Co.)

It was observed that a new band appeared at 245 nm and the CD spectrumintensity at 275 nm increased 1.5 times when the unmodified (natural)schizophyllan formed a complex with poly(C). With the 2.5%, 17% and 37%amino group-modified schizophyllans, there was observed a spectrum quitesimilar to that of the unmodified schizophyllan-poly(C) complex althoughthe intensity at 245 nm slightly increased. These spectral observationswere highly reproducible suggesting that the spectral change is not dueto unspecific adsorption based on cationic, electrostatic interactionsand that the 2.5%, 17% and 37% amino group-modified schizophyllans formsa complex with poly(C) in a similar manner to the unmodified (natural)schizophyllan. With polyethyleneimine, there was observed a decrease inthe CD spectrum intensity.

The stoichiometric analysis of the complex was made through UVabsorption measurement: The 17% amino group-modified schizophyllan wasdissolved in dimethyl sulfoxide to give a final concentration of 0.5g/dL. The solution of the single-stranded polysaccharide thus obtainedwas added to 0.1 g/dL poly(dA) solution in 100 μL pure water to preparesolutions with varying molar ratios of the nucleic acid to theschizophyllan. Then 10 mM Tris buffer (pH 8.0) was added to make thenucleic acid concentration in the solution constant. The DMSO in thesystem was replaced by water through ultrafiltration. The disappearanceof the DMSO was confirmed by UV absorption measurement at 230 nm. It wasascertained, through a preliminary experiment to measure changes in theweights of the solute and the solvent, that there occurred no change inthe nucleic acid concentration during the process.

The samples were measured for the ultraviolet absorption spectra. Theformation of the complex was examined through the hypochromic effect inthe UV absorption (decrease in the absorbance due to the formation ofthe complex). The results are shown in FIG. 7, and demonstrate thatthere was formed a complex composed of 2 moles of the 6-1,3-glucan and 3moles of the nucleic acid in terms of the number of moles of therepeating units (cf. the lower part of FIG. 7), i.e., that the complexwas composed of the double-stranded polysaccharide plus thesingle-stranded nucleic acid.

Example 4 Melting Behaviors of Unmodified Schizophyllan and AminoGroup-Modified Schizophyllan

The solutions prepared in Example 3 and Comparative Example 1 weremeasured for the temperature dependence of the CD spectrum. FIG. 9illustrates the data for the CD spectra at 275 nm in terms of molecularellipticity plotted against the temperature. ● denotes data for thepoly(C) alone, ▪ for the poly(C) plus the unmodified schizophyllan, ▴for the poly(C) plus the 2.5% amino group-modified schizophyllan, ♦ thepoly(C) plus the 17% amino group-modified schizophyllan, and ▾ for thepoly(C) plus the 37% amino group-modified schizophyllan, respectively.

As can be seen from FIG. 9, the complexes of the amino group-modifiedschizophyllans with the poly(C) show melting behaviors similar to thecomplex of unmodified (natural) schizophyllan with the poly(C). It wasalso demonstrated that the modified schizophyllan-based complexes weremore stable in terms of the melting temperature (Tm) than the unmodifiedschizophyllan-based complex: by 7° C. for the 2.5% amino group-modifiedschizophyllan, 14° C. for the 17% amino group-modified schizophyllan,and as great as 20° C. for the 37% amino group-modified schizophyllan.

Example 5 Interaction of Amino Group-Modified Schizophyllan with VariousNucleic Acids

Study was made on the interaction of the 2.5% amino group-modifiedschizophyllan as prepared in Example 1 with various types of nucleicacids. The 2.5% amino group-modified schizophyllan was dissolved indimethyl sulfoxide to give a final concentration of 0.5 g/dL. To 100 μlof the solution thus prepared, there were added 900 μl pure water, 100μl of 10 mM Tris buffer (pH 8.0), and 100 μl of 0.1 g/dL nucleic acidsolution. The resultant solutions were all clear and homogeneous.

The homogeneous nucleic acids used were poly(A) (Pharmacia), poly(U)(Yamasa), poly(G) (Sigma), poly(dA) (Pharmacia), poly(dT) (Pharamacia),poly(dC) (Pharmacia), and poly(dG) (Synthesized solid product). Anantisense DNA was also used that was composed a sequence ofCTTTAAGAAGGAGATATACAT (SEQ ID NO:1), with the 3′ end thereof beinglinked to forty dA's. The antisense DNA contained a sequencecomplementary to the sequence of GAAATTCTTCCTCTATATGTA (sequence for thelysome binding site of T7 promoter carried by T7 phage).

These nucleic acids were measured for the melting curve in the samemanner as in Example 4. With all the nucleic acids, the spectral curvefor the 2.5% amino group-modified schizophyllan is quite similar to thatfor the unmodified schizophyllan except for an observed increase in themelting temperature in the former. The values for melting temperatureobtained from the melting curves are shown in Table 2.

TABLE 2 Melting temperature (Tm) Nucleic Unmodified 2.5% aminogroup-modified acid schizophyllan schizophyllan Δ Tm poly(A) 30° C. 33°C. 3° C. poly(U) * 17° C. poly(G) * * poly(dA) 70° C. 80° C. 10° C. poly(dC) * 17° C. poly(dT) 16° C. 18° C. 2° C. poly(dG) * * Antisense33° C. 38° C. 5° C. * No complex formation

Example 6 and Comparative Examples 3 & 4 Antisense Effect Test

With the 2.5% amino group-modified schizophyllan as prepared in Example1 (this modified schizophyllan of the present invention is sometimesreferred to as SPG in this Example and Examples 7 through 10), a testfor antisense effect was conducted using a well-known cell-free systemin which transcription and translation reactions proceed in a cellularextract from E. Coli T7 S30. As an indication of antisense effect, atemplate was employed composed of a plasmid pQBI63 (Takara Shuzo)encoding GFP (Green Fluorescence Protein) which is a fluorescent proteinand widely used as a reporter gene. For the transcription andtranslation reactions, there was used E. coli T7 S30 in-vitrotranscription/translation kit (Promega). The antisense chain contained asequence CTTTAAGAAGGAGATATACAT (SEQ ID NO:1), as describe in Example 5,with the 3′ end thereof being linked to forty dAs. To the antisense DNAwas added the modified schizophyllan of the present invention (SPG) inan amount of 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, or 5.0-fold on a molarbasis, in the manner described in Example 5. The resultant solutionswere matured in a refrigerator for five days, and then subjected toultrafiltration with a membrane having an exclusion limit of 3000 toremove the dimethyl sulfoxide (Example 6).

To the antisense DNA was added polyethyleneimine (PEI) in an amount of0.1, 0.5, 1.0, 5, 10, or 50-fold on a molar basis, for the purpose ofcomparison (Comparative Example 3). For the purpose of furthercomparison, the influence of the modified schizophyllan alone was alsostudied with schizophyllan samples prepared by dialyzing (in distilledwater) the schizophyllan as used in Example 5 with a membrane having anexclusion limit of 12000 (Comparative Example 4).

The reaction solutions were prepared by adding the antisense DNA, theSPG complexes, the PEI complexes, or the schizophyllan samples to themixture containing the template DNA and the transcription/translationsolution. Each of the reaction solutions was allowed to undergo thetranscription and translation reactions by being kept at 37° C.,followed by the measurement of fluorescence emitted by GFP in thereaction solution (with a device from Hitachi). The data were normalizedin such way that the fluorescence intensity at 507 nm with the solutionnot containing the antisense DNA was 100.

The results are shown in FIG. 10. As can be seen from the figure, theaddition of the antisense DNA resulted in a decrease in the intensity ofthe fluorescence due to the expression of GFP. In addition, thetranscription and translation of GFP reporter gene were more suppressedwith increasing amount of SPG added to the antisense DNA. It is believedthat such antisense effect is reliably achieved through the release ofthe antisense DNA from the antisense DNA-SPG complex in which theantisense DNA is protected by SPG. It is noted that the addition of SPGin an amount of two-fold or more did not produce any further suppressioneffect. This is presumably because the formation of the complex proceedson a stoichiometric basis and an addition exceeding thestoichiometrically needed amount does not provide any further effect orbecause an excess addition may result in a decrease in the solubility.

By contrast, the addition of PEI did not achieve any substantial effectover the case where PEI was not added, and even an increase in theamount thereof did not result in any enhanced suppression. It is thusevidenced that the use of the polysaccharide of the present invention isquite advantageous. It is further noted that the excess addition of PEIlowered the suppression effect. This is presumably because the excesscationic charges on PEI resulted in insolubilization of the complex,lowering of the releasability or non-specific binding due to theelectrostatic interaction. Equimolar or excess addition of theschizophyllan to the antisense DNA caused substantially no decrease inthe fluorescence intensity, suggesting that the schizophyllan aloneexerted almost no influence on the transcription and translation of GFPreporter gene.

Example 7 Change in Antisense Effect with the Elapse of Time

The solution of the schizophyllan in dimethyl sulfoxide was added to thesolution containing an antisense DNA having a sequence ofCTTTAAGAAGGAGATATACAT (SEQ ID NO:1) with the 3′ end thereof being linkedto forty dA's as used in Examples 5 & 6 (referred to as antisense DNA),or another antisense DNA having a sequence of CTTTAAGAAGGAGATATACAT (SEQID NO:1) with each end thereof being linked to forty dA's (referred toas antisense DNA-2). Specifically, the modified schizophyllan was addedto each antisense DNA's in an amount of 1.0 fold on a molar basis, inthe manner of Example 5. The resultants were matured in a refrigeratorfor five days, and then subjected to ultrafiltration with a membrane(having an exclusion limit of 3000) to remove the dimethyl sulfoxide soas to form the complexes (referred to as +SPG complexes). The testingfor antisense effect was performed in the same manner as in Example 6.Thus, each reaction solution was prepared by adding each of the DNA's oreach of the SPG complexes to the mixture containing the template DNA andthe transcription/translation solution. The transcription andtranslation reaction was carried out, while measuring fluorescenceintensity due to GFP after 0, 0.5, 1.0, and 3.0 hours after theinitiation of the reaction. The data were normalized in such way thatthe fluorescence intensity at 507 nm after the 3.0 hour with the case ofno DNA addition was 100.

The results are given in FIG. 11. As can be seen from the figure, afterthe elapse of 30 minutes from the initiation of the reaction, +SPGcomplexes showed decreased fluorescence intensities at 507 nn due to theexpression of GFP, as compared with the references, suggesting that theformer enhance the suppression of the GFP expression. No substantialchange was observed in the antisense effect up to the three hours. It isalso noted that there was no difference in the antisense effect betweenthe termini of the antisense chains to which the dA's were linked.

Example 8 Evaluation of Resistance to Nuclease by Complex

The solution of the modified schizophyllan of the present invention indimethyl sulfoxide was added to the buffer solution containing theantisense DNA as described Examples 5 and 6. The mixture was matured ina refrigerator for five days, and then subjected to ultrafiltration witha membrane (having an exclusion limit of 3000) to remove the dimethylsulfoxide. To the resultant was added S1 Nuclase (Takara Shuzo), anuclease for specifically decomposing single-stranded nucleic acid, inan amount of 3 U so that the final concentration of the schizophyllanwas 6.1×10⁻⁴M, and that of zinc sulfate was 10 mM. Then, thedecomposition of the antisense DNA, in the presence of the schizophyllan(referred to as +SPG), was followed by measuring the absorbance at 260nm, due to the nucleic acid on a spectrophotometer (Jasco). Themeasurements were also made under conditions where no schizophyllan waspresent (as reference).

The results are shown in FIG. 12. The degree of increase in theabsorbance at 260 nm was clearly smaller in the case of +SPG as comparedwith the reference. This is presumably because the formation of theantisense DNA-SPG complex brought about resistance to the nuclease,thereby resulting in an enhanced antisense effect as in Example 7.

Example 9 Enhanced Antisense Effect Due to Resistance to Nuclease

The modified schizophyllan of the present invention in dimethylsulfoxide was added to the solution containing the antisense DNA asdescribed in Example 5 and 6. Specifically, the modified schizophyllanwas added to the antisense DNA in an amount of 1.0 fold on a molarbasis, in the manner described in Example 5. The resultant was maturedfor five days in a refrigerator, and then subjected to ultrafiltrationwith a membrane (having an exclusion limit of 3000) to remove thedimethyl sulfoxide so as to form the complex (referred to as +SPGcomplex). The antisense effects were evaluated in the same manner as inExamples 6 and 7. Thus, a reaction solution was prepared by adding theantisense DNA (as reference) or the SPG complex to the mixturecontaining the template DNA, the transcription/translation solution andmagnesium chloride. To each of the reaction solutions thus prepared wasadded Exonuclease I (Pharmacia), a nuclease for single-stranded DNA, inan amount of 1 U. Then the transcription and translation reaction wereperformed at 37° C., followed by measurement of fluorescence due to GFP.The measurement of fluorescence was also made with the sample notcontaining the antisense DNA, with the fluorescence intensity thereofbeing normalized as 100 (as reference).

The results are shown in FIG. 13. As can be seen from the figure, theSPG complex showed a decreased intensity in the fluorescence at 507 nmdue to the expression of GFP, as compared with the reference, in whichthe difference therebetween was larger than that observed in Example 6.It was thus evidenced that the complex will serve to suppressefficiently the GFP expression.

Example 10 Association of Complex and Substitution with ComplementaryStrand

In a similar manner to that described in Example 3, there was prepared amixture (1200 μl) of the modified schizophyllan of the present inventionand poly (dT). In sodium chloride solutions in water with varyingconcentration in the range of 0.05M to 0.5M, there were dissolvedpoly(dA) in equimolar amounts with poly(dT) contained in theabove-prepared mixture. Each of the poly(dA) solutions was addeddropwise to said mixture containing poly(dT). On measuring the resultantsolutions for the CD spectra after three hours while being kept at 10°C., there were observed well-known CD spectra due to thepoly(dT)-poly(dT) double helix. Ultraviolet absorption spectrummeasurements also gave an absorption coefficient attributable to atypical double-strand poly(dA)-poly(dT) complex (cf. ExperimentalChemistry Series, Nucleic Acid II). Thus, the modifiedschizophyllan-poly(dT) complex of the present invention was decomposedby the presence of poly(dA), leading to the formation of a new complex,the poly(dA)-poly(dT) complex. This example illustrates that thepresence of a complementary strand causes a rapid hybridization.

Example 11 Synthesis of Cholesterol-Modified Schizophyllan

The hydroxyl group of cholesterol (1 mg) was allowed to react withexcess phosgene (triphosgene) to chloroformate ester (yield: 80%). Thechloroformate ester-substituted cholesterol was then allowed to reactwith equimolar ethylene diamine. The cholesterol derivative thusprepared and containing an amino group was introduced into thesingle-stranded schizophyllan (s-SPG) in the manner described inExample 1. The rate of introduction was regulated through the amounts ofthe starting cholesterol derivative, which can be determined byelemental analysis. There were obtained cholesterol-introducedschizophyllans with the rate of introduction being 2 mol %, 10 mol % and30 mol %, which are referred to as C-s-SPG-O₂, C-s-SPG-10 andC-s-SPG-30, respectively.

Example 12 Synthesis of Peptide-Modified Schizophyllan

As a example of amino acid-based functional group, integrin-bindingpeptide (cystein-tyrosine-glycine-glycine-arginine-glycine-asparticacid-threonine-proline) was introduced into the schizophyllan.Specifically, 1 mg of the peptide, with the C-terminus thereof beingprotected by an esterification, and the triple helix schizophyllanfollowing the periodate oxidation as described in Example 1, were addedto 20 mL DMSO, followed by stirring for an hour at room temperature. Theresultant was then subjected to treatment with a large amount of sodiumborohydride, reprecipitation with methanol, and lyophilization. Thesample thus obtained is herein referred to as InG-s-SPG.

Example 13 Synthesis of Intercalator-Modified Schizophyllan

The schizophyllan was chemically modified with an acridine derivative:In accordance with the reaction scheme illustrated in FIG. 6, anaminoalkane carboxylic acid (the one shown in FIG. 6 in which R_(x) isC₂H₄), as the spacer moiety, was introduced into acridine, usingdicylohexyl carbodiimide as the condensation agent. The aminatedacridine thus obtained was subjected to the reductive aminationreaction, as in Example 1, together with the single-strandedschizophyllan following the periodate oxidation. The sample thusprepared is herein referred to as Ac-s-SPG. Elemental analysis showedthat the rate of introduction was 6%.

Example 14 Confirmation of Complex Formation

The formation of complexes of the modified schizophyllan, as prepared inExample 11 and 13, with nucleic acids was examined through CD spectrummeasurement, the fluorescence depolarization method, and gelelectrophoresis, An outline of the conditions and methods for theexperiments follows:

The measurements of CD spectra were carried out in the same manner as inComparative Example 2. The fluorescence depolarization method wascarried out on JACSFP-715. Marking of the modified schizophyllans with afluorochrome was conducted in the manner described in Example 15. Thesolutions were diluted for the fluorescence measurements and the complexformation was judged at the point where the P value indicating thedegree of fluorescence depolarization was 50% of that of theschizophyllan marked with the fluorochrome. The gel electrophoresis wasexamined with agarose gel, each gel being stained with gelstar (FMC:Bioproduct) following a three-hour migration. Almost no migration occursupon the formation of a complex, whereas free nucleic acids continue tomigrate. The results are summarized in Table 3.

TABLE 3 Single-stranded DNA as described in Example 5 but DNA fromPoly(C) having no poly(dA) tail salmon's sperm C-s-SPG-02 ◯ ◯ X-ΔC-s-SPG-10 ◯ ◯ ◯ C-s-SPG-30 ◯ ◯ ◯ Ac-s-SPG ◯ ◯ ◯ Comparative: ◯ X XUnmodified single-stranded schizophyllan ◯ Complex formation X Nocomplex formation

Example 15 Introduction of Complexes into Cells

Hep G2 cells, human hepatic cells (available from American Type CultureCollection: ATCC), were cultured in Eagle's minimum essential medium(EMEM medium: Sigma) supplemented with 10% fetal bovine serum (FBS:Wako) at 37° C. with 5% CO₂, from which there was obtained Hep G2 cellculture supernatant. InG-s-SPG, as prepared in Example 12, and s-SPGwere each dissolved in anhydrous DMSO and the resultant solutions wereeach added with a small quantity of fluoresceine isocyanate to providethe saccharide chains with a fluorescence marker. The samples thusprepared are herein referred to as InG-s-SPG-FITC and s-SPG-FITC,respectively. The antisense DNA used in Example 6, CTTTAAGAAGGAGATATACAT(SEQ ID NO:1)-(dA)40, was modified at the 3′end thereof, with rhodamine,as a marker. The DNA sample thus prepared is herein referred to asR-DNA1.

R-DNA1 plus InG-s-SPG-FITC and R-DNA1 plus s-SPG-FITC were eachdissolved in a DMSO solution. In each case the polysaccharide was addedto 1.5 times excess of the stoichiometric amount: This reliably formedthe complexes and it was confirmed that the excess polysaccharide notused for the complex formation did not at all adversely affect thesystem. The formation of the complexes was monitored by gelelectrophoresis. The resultant samples were each added to the Hep G2cell culture supernatant, followed by culturing at 37° C. with 5% CO₂.

At four hours after the initial contact of the samples with the cells,the culture medium was discarded, followed by washing the cultures witha phosphate buffer and fixation of the cells with 4% paraformaldehyde.The resultants were observed under a fluorescence microscope (OlympusIX70-22PH). As a result, fluorescent cells stained with fluoresceine inthe case of the addition of InG-s-SPG-FITC were observed at 10 to 100times the number observed in the case of the addition of s-SPG-FITC, nopeptide-modified sample. Monitoring of the degree of the antisense DNAtransferred into the cells through the rhodamine coloration alsoindicated that the addition of InG-s-SPG-FITC was 10 to 100 times higherthat in the case of the addition of s-SPG-FITC, no peptide-modifiedsample.

Example 16 Introduction of Complexes into Cells

C32 cells, human melanoma cells (available from ATCC), were cultured inEMEM medium supplemented with 10% FBS and 10% nonessential amino acidsat 37° C. with 5% CO₂. The resultant thus obtained is herein referred toas C32 culture cell supernatant. The 17% amino group-modified cationicschizophyllan prepared in Example 1 (herein referred to as N-s-SPG17)was provided with the fluorescence marker in the same manner as inExample 15. The resultant thus obtained is herein referred to asN-s-SPG17-FITC. R-DNA1, the rhodamine-modified nucleic acid sample, wasprepared in the same manner as in Example 15, and a complex ofN-s-SPG17-FITC and R-DNA1 was formed. The complex was added to C32 cellculture supernatant, followed by culturing at 37° C. with 5% CO₂. Atthree hours after the initial contact of N-s-SPG17-FITC with the cells,the culture was subjected to cell fixation for examination under afluorescence microscope. There was observed fluorescence emission on thecell surfaces, from which it was confirmed that N-s-SPG17-FITC wasintroduced into the cells. In addition, the rhodamine colorationindicated that the degree of the antisense DNA transferred into thecells in the case of the addition of N-s-SPG-FITC was 10 to 100 timeslarger than that in the case of the addition of s-SPG-FITC, non-modifiedsample.

SEQUENCE LISTING

-   <110> Japan Science and Technology Corporation,-   <110> KIMURA Taro-   <110>MIZU Masamui-   <120> Gene carrier utilizing polysaccharide and method for making    same-   <130> P0266T-PCT-   <150> JP P2001-069655-   <151> 2001-03-13-   <150> JP P2001-130705-   <151> 2001-04-27-   <160> 1-   <210> 521-   <211> 21-   <212> DNA-   <213> Artificial Sequence-   <400 > 1-   ctttaagaag gagatataca t

1. A gene carrier composed of a complex in which a single-strandednucleic acid is bound to a double-stranded β-1,3-glucan wherein theβ-1,3-glucan has at least one 1,6-glucopyranoside branch per repeatingunit of polysaccharide and is selected from schizophyllan, lentinan,grifolan, and scleroglucan, and wherein at least some of1,6-glucopyranoside branches are chemically modified to befunctionalized with nucleic acid-binding functional groups which arecationic functional groups formed by reaction of the polysaccharide withchain or cyclic compounds containing at least one primary or secondaryamino group, wherein the chemical modification is carried out byperiodate-oxidizing the 1,6-glucopyranoside branches and thenreductive-aminating the periodated 1,6-glucopyranoside branches toimpart the nucleic acid binding functional group thereto, and whereinthe 1,6-glucopyranoside branch which has been functionalized with thenucleic acid-binding groups is expressed by the following generalformula (1):

wherein the two X's are the cationic functional groups and are identicalor different.
 2. The gene carrier of claim 1, wherein the nucleicacid-binding functional groups are introduced at a rate of at least 0.1mol % and up to 50 mole % based on the repeating unit of thepolysaccharide.
 3. The gene carrier of claim 1, wherein the β-1,3-glucanhas a molecular weight of at least
 2000. 4. The gene carrier of claim 1,wherein the nucleic acid is an antisense DNA.
 5. The gene carrier ofclaim 1, wherein the β-1,3-glucan comprises schizophyllan.